Neutrophils promote inflammatory angiogenesis via release of preformed VEGF in an in vivo corneal model

Abstract

We investigated the role of neutrophilic cells (neutrophils) in inflammatory angiogenesis and explored the possible mechanisms involved. Corneal angiogenesis was induced in vivo with a 75% silver nitrate applicator. Depletion of neutrophils was accomplished by the intraperitoneal administration of RB6-8C5, a neutrophil-depleting antibody. Angiogenesis, neutrophil infiltration, and the localization of vascular endothelial growth factor (VEGF) were evaluated by biomicroscopic observations, histology, and immunohistochemistry in control and RB6-8C5 treatment groups. Protein levels of VEGF, macrophage inflammatory protein-1alpha (MIP-1alpha), macrophage inflammatory protein-2 (MIP-2), and tumor necrosis factor alpha in the cornea were determined by enzyme-linked immunosorbent assay. An in vitro model of neutrophil activation was also used to examine the ability of neutrophils to produce and release VEGF, MIP-1alpha, and MIP-2. At day 1 after injury, neutrophil infiltration in the cornea was highest, and VEGF was expressed in the infiltrating neutrophils. The enhanced protein levels of VEGF, MIP-1alpha, and MIP-2 correlated with the degree of neutrophil infiltration. Neutrophil depletion significantly inhibited corneal angiogenesis and reduced the protein levels of VEGF, MIP-1alpha, and MIP-2 in the cornea. Upon stimulation, isolated neutrophils released VEGF from preformed stores and MIP-1alpha and MIP-2 by de novo synthesis. Neutrophil depletion thus significantly impaired inflammatory angiogenesis, identifying neutrophils as an important player in inflammatory angiogenesis. Neutrophils may exercise their angiogenic function by releasing proangiogenic factors such as VEGF. Intervention measures targeting neutrophils may therefore help to deal with abnormal angiogenesis involved in chronic inflammatory diseases.

Early effects of dexamethasone and anti-VEGF therapy in an inflammatory corneal neovascularization model

“…The model, designed for longitudinal in vivo examination, induces vessels in a well-defined region of the cornea between the limbus and the suture, limited to about one-tenth of the cornea's total surface area. In other animal models with several central sutures (Bock et al 2007;Cursiefen et al, 2004;Hos et al, 2011) or a large-area alkali burn (Dratviman-Storobinsky et al, 2009;Gong and Koh, 2010;Hoffart et al, 2010;Sener et al, 2011) a large region of the cornea can become vascularized, which allows for greater sensitivity in detecting significant reductions in invasion area, but a less predictable and controlled pattern of vascularization.…”

“…For example, stromal invasion of leukocytes (Bourghardt Peebo et al, 2007;Fagerholm and Gan, 2001;Gong and Koh, 2010;Nakao et al, 2012) which produce pro-angiogenic cytokines, precede and promote vessel invasion, and at a later stage could reactivate vessel growth upon cessation of treatment (Cursiefen et al, 2004;Gong and Koh, 2010;Lu et al, 2012;Sakurai et al, 2003;Sunderkötter et al, 1994;Tazzyman et al, 2013). Besides this paracrine function, evidence is mounting that inflammatory cells may have a more direct role in tissue remodeling and the formation of new vessels (Bourghardt Peebo et al, 2011;Fantin et al, 2010;Kim et al, 2009;Maruyama et al, 2005).…”

Intravascular Leukocyte Chemotaxis: The Rules of Attraction

“…The neutrophils are in fact the only cells in the body that release MMP-9 free of its endogenous inhibitor TIMP (tissue inhibitor of metalloproteinase), and are therefore capable of deliver highly active MMP-9 to sites of angiogenesis (Ardi V. C. et al, 2007). The pro-angiogenic capacity of neutrophils has been demonstrated in a corneal injury model, where the number of infiltrated neutrophils positively correlated to angiogenesis and VEGF levels (Gong Y., Koh D. R., 2010). Neutrophil depletion significantly impaired tissue healing in this model, as well as the release of VEGF.…”

Revisiting the protective and pathogenic roles of neutrophils: Ly‐6G is key!

“…Neutrophils are the most abundant leukocytes at inflammatory sites and, therefore, much attention has been placed on ascertaining the role they play in inflammation and how they initiate and regulate innate and adaptive immune responses during microbial infection [1][2][3]. Neutrophils are short-lived myeloid cells that cannot easily be manipulated genetically and no neutrophil knockout animals have been successfully established.…”

“…Furthermore, the findings [13][14][15] remind us that all previous studies that used the anti-Gr-1 mAb (clone RB6-8C5) to investigate the roles of neutrophils need to be revisited and their conclusions reconsidered. Neutrophils have been implicated in the pathogenesis of many kinds of diseases, with both inflammation-initiating (harmful) [3,16,17] and antiinflammatory (protective) [4,5] functions being reported; however, the mechanisms that modulate this plasticity of neutrophils remain unclear. Neutrophils have been found to be important in the pathogenesis of inflammatory autoimmune diseases [16,17].…”